Brain damage including focal and global cerebral injury as well as suboptimal cognitive developmental outcome continue to be important problems after pediatric heart surgery. Previous work in this area has focused on deep hypothermic circulatory arrest (DHCA) which is now used infrequently. As an alternative to DHCA reduced flow hypothermic cardiopulmonary bypass (CPB) is employed. However in the absence of a validated method for real time monitoring of brain oxygenation there are no guidelines for minimal safe flow and pressure under specific CPB conditions of pH, hematocrit and temperature. The proposed study will employ the new techniques of near infrared spectroscopy (NIRS) and intravital microscopy (IVM) to defme a minimal safe flow rate for specific perfusion conditions. The study will be conducted using a juvenile piglet model exposed to various degrees of flow reduction with survival for 4 days postoperatively. Survival allows assessment of functional evidence of brain injury through behavioral assessment by a blinded veterinarian observer as well as meaningful histology determined by a blinded neuropathologist. These functional and structural endpoints will be correlated with indices of brain oxygenation measured by NIRS (Tissue Oxygenation Index (TOl), Oxyhemoglobin nadir time (Hb02 nadir time)) as well as indices of microvascular perfusion measured by IVM (functional capillary density (FCD), NADH fluorescence). The second phase of the proposed study will test the hypothesis that critically reduced low flow perfusion causes hypoxic endothelial injury of cerebral blood vessels. This results in reduced constitutive endothelial nitric oxide synthase (eNOS) activity resulting in microvascular regional ischemia previously described as the "no reflow phenomenon." Acute studies will be undertaken in the piglet model using Western immunoblotting and immunocytochemistry as well as resistance vessel myography to measure eNOS activity. eNOS activity will be manipulated by substrate enhancement and inhibition. The role of inducible NOS (iNOS) in causing neurotoxicity in this setting will also be explored. The proposed study has the potential to reduce the risk of brain injury in children undergoing heart surgery by defining the margin of safety achieved with various perfusion conditions. By enhancing understanding of mechanisms of cardiopulmonary bypass-related brain injury it will facilitate development of novel pharmacologic methods to further reduce the risk of brain damage.